Microfluidic PCR is generally defined as PCR in chambers with the dimensions being very small, for example between a range of 500 and 0.5 μm. There are microfluidic systems with various heating systems such as zone heating, external heaters, integrated resistors, Joule heating, and infrared radiation, but those systems require external detection. That means that at the end of the reaction, some of the sample has to be physically removed from the PCR chamber and analyzed externally from the system by means such as gel electrophoresis or capillary electrophoresis. Examples of work of this type are described in Cheng et al. Nucleic Acids Res, 24(2), p. 380-5 (1996); Kopp and Manz, Science, 280(5366) p. 1046-8 (1998); Oda Anal Chem, 70(20), p. 4361-8 (1998); and Chen et al. Anal Chem, 77(2): p. 658-66 (2005). A system requiring external detection would be incompatible with real-time detection functions because such a system would require removal of a large part of the PCR reaction solution for external measurements.
Another detection method uses optical detection, which involves intercalating dyes such as SYBR Green (see Rasmussen et al., Biochemica, 1998(2): p. 8-111998), or fluorogenic probes such as Taqman probes (see Kalinina, O., et al., Nucleic Acids Res, 25(10), p. 1999-2004 (1997); Belgrader, P., et al., Science, 284(5413), p. 449-50 (1999); Belgrader, P., et al. Anal Chem, 75(14), p. 3446-50 (2003); and Northrup, M. A., et al., Anal Chem, 70(5), p. 918-22 (1998)). SYBR Green dye is a highly specific, double-stranded DNA binding dye for detecting DNA product as it accumulates during PCR cycles. Although both detection methods can be used in a real-time PCR amplification, a distinction between the intercalating dye and the Taqman probe techniques is that the intercalating dye detection method is not specific. It will detect all double-stranded DNA, including non-specific PCR products such as primer-dimer formations. For this reason PCR amplification with an intercalating dye often requires a melting curve analysis to distinguish between a desired PCR product and non-specific PCR amplification.
In contrast, the Taqman probe PCR detection method is highly specific because the generation of fluorescent signal depends on the specific hybridization between the probe and the desired PCR product. However, the Taqman probe PCR detection method requires, unlike SYBR Green dye, synthesizing individual probes for different sequences, is constrained by the limitation of fluorescent dyes that can be used for probe labeling (limited by the number of different color dyes, for example), and needs expensive reagents.
An alternative detection method for PCR is to perform an electrophoretic DNA size separation of the reaction products. This allows the desired reaction product to be identified separately from all other sources of DNA or contaminants. Many microfluidic PCR systems with external detection involve DNA sizing, either on a gel or by capillary electrophoresis, because it is specific and does not require target-specific probe synthesis.